GC-rich DNA sequences are found within the promoter and first exon of approximately 50% of all genes in the human genome (Antequera & Bird, Proc. Natl. Acad. Sci. USA, 90:11995-11999, 1993). These sequences are known in the art as CpG islands. CpG islands can be targets of DNA methylation, an epigenetic phenomenon associated with altered chromatin structure and transcriptional repression (Rountree, M. R. et al., Oncogene, 20:3156-3165, 2001). Mammalian cells possess methylases that methylate cytosine residues on DNA that are 5′ neighbors of guanine in CpG dinucleotides (CpG). Methylation occurs after cytosine has been incorporated into DNA in a process catalyzed by DNA methyltransferases (“Dnmts”), which transfer the methyl group from S-adenosylmethionine to the 5′-position of the pyrimidine ring in, characteristically but not exclusively, the context of the palindromic CpG dinucleotide (Ramsahoye et al., 2000). 5-Methylcytosine is asymmetrically distributed in the genome and is most commonly found in CpG-poor regions, since most CpG islands in somatic cells remain methylation-free, except for the promoters of imprinted genes and genes on the inactive X-chromosome (Bird et al., 1985) where methylation of 5′ regulatory regions can lead to transcriptional repression.
Thus, in normal cells, CpG island methylation plays an important role in regulating gene expression in a developmentally significant and tissue-specific manner (Jones & Takai, Science, 293:1068-1070, 2001). In cancer cells, however, aberrant methylation (typically hyper-methylation) of CpG islands has been found in the 5′-end of the regulatory region of many tumor-suppressor genes and of genes responsible for genomic stability (Esteller, M. et al., Cancer Res., 61:3225-3229, 2001; Baylin, S. B. et al., Hum. Mol. Genet., 10:687-692, 2001; Jones & Laird, Nat. Genet., 21:163-167, 1999). Current data is consistent with a causal link between his type of epigenetic control and tumor development (Jones & Laird, 1999). Accordingly, critical tumor-suppressor and stability genes are silenced, leading to clonal proliferation of tumor cells (e.g., Id).
Thus, hypermethylated CpG islands that can be associated with gene silencing provide at least potential markers for novel genes that are subject to epigenetic control, and thereby have potential important diagnostic and prognostic utility. However, efficient identification in such associated cases of a direct or primary (causative) association of methylation and gene silencing is problematic, because many genes participating in cell-cycle control, growth factor/receptor signaling, and mobilization of retroelements appear to be generally up-regulated in a demethylated cellular state (Jackson-Grusby, L. et al., Nat. Genet., 27:31-39, 2001), and such regulation is likely to be secondary, or indirectly related to CpG-island methylation as part of a downstream, epigenetic expression cascade.
Therefore, prior art methylation assays, alone or in combination with prior art gene expression assay methods do not provide efficient and accurate methods for identification of novel direct associations between methylation and gene silencing. This is because divergent sets of reagents (e.g., methylation probes, and cDNA probes) would be required in independent assay procedures (e.g., methylation assays, and Northern blots or cDNA array screens), followed, if possible, by careful data correlation.
For example, there are various art-recognized assays for assessing the methylation state at particular CpG sequences, once the sequence region comprising them has been identified so that specific primers and/or probes can be constructed. Such assays include: DNA sequencing methods; Southern blotting methods; MethyLight™ (fluorescence-based real-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999; U.S. Pat. No. 6,331,393); MS-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension assay described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997; U.S. Pat. No. 6,251,594); MSP (Methylation-specific PCR assay described by Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146); and COBRA (Combined Bisulfite Restriction Analysis methylation assay described by Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).
Likewise, assays for the discovery of novel differentially methylated CpG sequences, while less numerous, include such methods as: restriction landmark genomic scanning (“RLGS”; Eng et al., Nature Genetics 25:101-102, 2000; Costello et al., Nature Genetics 25:132-138, 2000; Zhu et al., Proc. Natl. Acad. Sci. USA 96:8058-8063, 1999); RLGS in combination with virtual genome scans (“VGS”; Rouillard et al., Genome Research 11:1453-1459, 2001); methylated CpG island amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-2312, 1999; WO 00/26401A1); arbitrarily primed-polymerase chain reaction (“AP-PCR”; Liang et al., Genomics 53:260-268, 1998); and differential methylation hybridization (“DMH”; Yan et al., Clin. Canc. Res. 6:1432-1438, 2000).
Restriction Landmark Genomic Scanning. Restriction landmark genomic scanning (“RLGS”) approaches have been employed to identify sequences and regions of differential methylation, and regions so-identified have been cloned and sequenced. RLGS methods take advantage of the fact that specific DNA cleavage by particular restriction enyzmes, such as NotI is methylation sensitive. Moreover, NotI has a CG-rich octanucleotide recognition motif, and cleaves predominantly in CpG-rich “islands.” Thus, digestion of genomic DNA with NotI and end-labeling of the NotI staggered ends, followed by further restriction digestion (e.g., with 5-base and/or 6-base recognition sequence enzymes) in combination with 2-dimensional electrophoresis has been used to generate resolved patterns of CpG-island-related fragments having at least one labeled NotI end. Such patterns can be used to compare the methylation status among various genomic DNA samples, and if a particular NotI site is methylated in a test genomic DNA sample, relative to that in normal genomic DNA, no corresponding end labeled fragment(s) will be visible in the RLGS pattern of the test sample (corresponding ‘spot disappearance,’ or absence). Boundary libraries (e.g., of NotI-EcoRV fragments) can be used to obtain cloned DNA corresponding to such regions.
Significantly, however, such prior art RLGS methods for detection of CpG methylation are limited, inter alia, by: (i) the use of only particular methylation-sensitive restriction enzymes, which effectively limits analyses to CpG sequences within CpG island regions; (ii) dependence (for detection) upon NotI end-labeling (or the equivalent); and (iii) upon the disappearance of (more accurately, the absence of) a test DNA spot (i.e., where a particular NotI site in a test DNA sample is methylated and therefore not cleaved by NotI digestion) relative to a corresponding spot present in the normal (test) DNA 2-dimensional pattern. Moreover, RLGS methods reveal nothing about gene expression, and therefore nothing about methylation-dependent gene silencing.
Virtual Genome Scans. Virtual genome scans (VGS) provide methods for use in conjunction with RGLS methods to identify fragments of interest displayed in RLGS scans. Informatics tools are used, in conjunction with known human genome sequence information, to produce virtual scans, for example, with NotI and EcoRV (as first-dimension RLGS restriction enzymes), and, for example, HinfI or DpnII (as second-dimension enzymes). The size of the expected NotI-EcoRV and NotI-NotI fragments (if no intervening EcoRV site is present) are computed, along with the second-dimension fragments, based on the HinfI or DpnII site nearest to a particular NotI site (Rouillard et al. Genome Research 11:1453-1459, 2001). Thus, identification of RLGS sequences can be made without the use of boundary libraries.
However, the method still depends on determining the differences between two samples using RLGS, and is thus subject to most of the limitations thereof. Moreover, as for RLGS, VGS reveals nothing about gene expression or methylation-dependent gene silencing.
Methylated CpG Island Amplification. Methylated CpG island amplification (“MCA”) is a PCR-based technique for rapid enrichment of hypermethylated CG-rich regions, that requires the sequential digestion by a particular methylation sensitive, methylation insensitive isoschizomeric enzyme pair (i.e., SmaI and XmaI, respectively), followed by PCR amplification based on primers that specifically hybridize to adapters ligated to the staggered XmaI ends. Additionally, the restriction sites must be closely situated (<1 kb apart).
Thus, as in the case of prior art RLGS applications, the method is primarily limited to particular CpG sequences within CpG-rich genomic regions (Toyota et al., Cancer Res. 59:2307-2312, 1999). Additionally, the technique is sensitive to artifacts relating to incomplete digestion with SmaI, the methylation sensitive restriction enzyme. The technique can be combined, in a more complex multistep method with substractive hybridization (RDA; representational difference analysis) to obtain cloned fragments enriched for hypermethylated sequences (Id). Nonetheless, as for RLGS and VGS methods, MCA reveals nothing about gene expression or methylation-dependent gene silencing.
Methylation-Sensitive Arbitrarily Primed PCR. Likewise, methylation-sensitive arbitrarily primed-polymerase chain reaction (“AP-PCR”) is a PCR-based technique for rapid enrichment of hypermethylated CG-rich regions, that involves co-digestion of DNA with a methylation-insensitive enzyme (e.g., RsaI) to generally reduce the size of DNA fragments, plus, in separate reactions, a methylation-sensitive member, and a methylation-insensitive member of a isoschizomeric enzyme pair (e.g., RsaI plus HpaII, and RsaI plus MspI, respectively), followed by PCR amplification using one or more specific oligonucleotide primers. In this case, no PCR products are produced if the region between two primer sites contains an unmethylated HpaII (CCGG) sequence. Digestion of the DNA with RsaI only, and with RsaI and MspI serve as controls for determining whether bands observed in the AP-PCR of RsaI- plus HpaII-digested DNA are actually due to differential methylation of CCGG sequences within the region of amplification (Conzalgo et al., Cancer Research 57:594-599).
Thus, methylation-sensitive AP-PCR methods are limited commensurate with primer choice, and as for RLGS and MCA described above, are primarily biased toward CpG island regions, especially when extensively CG-rich primer sequences are employed (Liang et al., Genomics 53:260-268, 1998). Generally, methylation-sensitive AP-PCR is subject to many of the same artifacts that limit the effectiveness of MCA methods, such as incomplete digestion by restriction enzymes, and distance between primer sites. Moreover as for RLGS, VGS, and MCA, AP-PCR reveals nothing about gene expression or methylation-dependent gene silencing.
Differential Methylation Hybridization. Differential methylation hybridization (“DMH”) is an array-based method involving differential probing of arrayed CpG-rich tags (e.g., from a CpG island genomic library) with amplicons from reference, or, e.g., tumor DNA samples (Huang, T. H.-M. et al., Hum. Mol. Genet., 8:459-470, 1999; see also applicant's U.S. Ser. No. 09/497,855, filed 4 Feb. 2000, Notice of Allowance received 7 Mar. 2003, and incorporated by reference herein in its entirety). The differences in tumor and reference signal intensities on the tested CpG island arrays reflect methylation alterations of corresponding sequences in the tumor DNA (Yan et al., Clin. Canc. Res. 6:1432-1438, 2000). Using a panel of CpG island tags arrayed on solid supports, DMH has been applied to identify hypermethylated genes in breast and ovarian cancer (Yan, P. S., et al., Clin. Cancer Res., 6:1432-1438, 2000; Ahluwalia, A. et al., Gynecol. Oncol., 82:261-268, 2001).
To produce DMH amplicons, the DNA is digested to produce small (e.g., about 200 bp) DNA fragments while preserving CpG islands (e.g., by digestion with MseI, which recognizes TTAA). Linkers are ligated to the fragment ends, and the fragments are digested with a methylation-sensitive enzyme, e.g., BstUI (77% of known CpG islands contain BstUI sites), prior to filling in the protruding linker ends and PCR amplification using linker primers. Fragments cleaved by the methylation-sensitive enzyme are rendered non-amplifiable by the linker primers, so that the amplified fragment pool is enriched for methylated amplicons.
DMF is thus favorably distinguished from other prior art methods as a high-throughput method for the analysis of CpG-rich genomic DNA regions. Currently, however, DMH is limited by the fact that only about 2% of the total genomic CpG island regions are represented in the available arrayed panels (Id). Moreover, as for all the prior art methods discussed above, including RLGS, VGS, MCA, and AP-PCR, the DMH protocol alone reveals nothing about gene expression and methylation-dependent gene silencing.
Demethylating Drugs and Gene Expression Assays. DNA methylation is unique in that it is a mechanism for modifying the base sequence of DNA without altering its coding, and because it is a heritable reversible epigenetic change. Currently, therefore, there is renewed enthusiasm for administration of demethylating drugs in cancer treatment (Santini, V., et al., Ann. Intl. Med., 134:573-86, 2001). Effective demethylating treatments, however, will require an understanding of how particular genes respond to particular demethylating agents in cancer cells. Specifically, as discussed above in relation to methylation assays and gene silencing, expression assays are needed, along with a method to efficiently correlate gene expression with gene methylation state.
Prior art gene expression assay methods include Northern blotting methods, RT-PCR-based methods, and cDNA-based screening methods. However, prior art expression assays reveal absolutely nothing about the methylation status of the subject genes, and significantly fail to distinguish between direct and indirect epigenetic effects. For example, cDNA microarrays have been applied to determine global profiles of gene expression in demethylated cells (Karpf, A. R. et al., Proc. Natl. Acad. Sci. USA, 96:14007-14012, 1999; Jackson-Grusby, L. et al., Nat. Genet., 27:31-39, 2001). However, such assays reveal nothing about the methylation state of the subject genes, and as discussed above, many genes participating in cell-cycle control, growth factor/receptor signaling, and mobilization of retroelements appear to be secondarily regulated as part of a down-stream epigenetic cascade, or could be regulated in diseased cell states independent of methylation.
Therefore prior art DNA expression methods cannot themselves be used to differentiate between or among genes that directly respond (primary response) to demethylation, and those indirectly-(secondary response) or independently-regulated genes.
Likewise, while DMH and other prior art methylation assays have great potential for identification of differentially methylated CpG sequences, they do not in themselves provide for an efficient method for the determination of whether the particular sequences so identified, are sequences that are transcriptionally regulated by the identified differential methylation.
Therefore, there is a need in the art for novel high-throughput methods that serve not only to identify and validate genes with hypermethylated promoters in neoplasia and other diseases, but also to simultaneously measure gene expression and thereby enable efficient identification of genes that are relevant to tumorigenesis or other aberrant cell functions. There is a need in the art for novel methods that can be utilized to efficiently study the efficacy, and optimal dosages of various types of demethylating agents in cancer treatment regimens.
Repressed chromatin and gene silencing. Microarray approaches used to study functional DNA-protein interactions (e.g., Ren et al., Science (Wash. DC) 290:2306-2300, 2000; Weinmann et al., Genes Dev., 16:235-244, 2002; and Suzuki et al., Nature Genet. 31:141-149, 2002) have revealed that many transcription regulators are linked to chromatin remodeling (Suzuki et al., Nature Genet. 31:141-149, 2002; and Cameron et al., Nature Genet. 21:103-107, 1999), placing this type of epigenetic change at the center of gene regulation. Specifically, repressed chromatin and gene silencing are associated with changes in DNA methylation and histone acetylation (Jones & Baylin, Nature Rev. Genet. 3:415-428, 2002), and these epigenomic modifications are widely recognized as a contributing factor in human tumorigenesis. Methylated DNAs at the 5′-end regulatory regions of genes recruit MBD (methyl-CpG binding domain) proteins, which are known to complex with histone deacetylases and other transcriptional corepressors (Ballestar & Esteller, Carcinogenesis 23:1102-1109, 2002). Deacetylation of lysine groups on histones 3 and 4 occurs via HDACs (histone deacetylases), resulting in a tighter interaction between negatively charged DNA and positively charged lysine and a closed, repressive chromatin configuration (Jones & Baylin, Nature Rev. Genet. 3:415-428, 2002; Ballestar & Esteller, Carcinogenesis 23:1102-1109, 2002). How repressive chromatin structures assemble onto DNA is not clear, but changes in methylation status of CpG islands in gene promoters presumably play a central role (Jones & Baylin, supra).
While DMH (differential methylation hybridization), as described above, is useful for screening CpG methylation and identifying loci susceptible to epigenetic modifications in various cancers, additional information is needed to characterize and elucidate the functional relationship between DNA methylation and histone acetylation in gene silencing.
There is a pronounced need in the art for a genomic microarray system for efficiently detecting changes in gene expression, DNA methylation and histone acetylation, and for distinguishing primary from secondary effects. There is a need in the art for such a genomic microarray system that is efficient therefore by virtue of that fact that all three of said parameters (gene expression, DNA methylation and histone acetylation) are assessed in parallel using a single microarray, or by using a plurality of the same microarray.